Process for producing cis-succinic acid of gauche form



1967 TADATAKA HARA PROCESS FOR PRODUCING CIS-SUCCINIC ACID OF GAUCHE FORM Filed NOV. 15, .1966

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INVENTOR BYM a M I ATTORNEY Nov. 14, 1967 TADATAKA HARA 3,352,759

PROCESS FOR PRODUCING CIS-SUCGINIC ACID OF GAUCHE FORM Filed Nov, 15, 1966 Absorbancy 4 Sheets-Sheet 2 Fumo'n'c ac/d (5/m Presenf subsfance (400/07! Succ/h/c 00/0 0 (6/076 0 1 l l 1 I l .200 2/0 220 Wave/engfh (mp) INVENTOR ATTORNEW Nov. 14, 1967 TADATAKA HARA 3,352,759

PROCESS FOR PRODUCING CIS-SUCCINIC ACID OF GAUCHE FORM Filed Nov. 15, 1966 Y 4 Sheets-Sheet 5 INV ENT OR ATTORNEYS Nov. 14, 1-967 TADATAKA HARA 3,352,759

rgocmss FOR PRODUCING bls-succmxc ACID OF GAUGHE FORM Filed Nov. 15, 1966 I 4 Sheets-Sheet 4.

United States Patent 3,352,759 PROCESS FUR PRODUCHNG ClS-SUCCINIC ACED 0F GAUCHE FORM Tadataka Ham, Tokyo, Japan, assignor to Kaken Kagaku Kabushiki Kaisha, Tokyo, Japan, a corporation of Japan Filed Nov. 15, 1966, Ser. No. 594,602 Claims priority, application Japan, Jan. 14, 1959, 34/534; June 6, 1959, 34/2tl,893 5 Claims. (Cl. 19537) This application is a continuation-in-part of my earlier application Ser. No. 860,227 filed on Dec. 17, 1959, now abandoned.

This invention relates to a novel and useful compound called cis-succinic acid of Gauche form particulary concerns a method for producing cis-succinic acid of Gauche form by fermentation and a method for deodorizing amine-containing materials by contacting them with the said cis-succinic acid of Gauche form.

Heretofore, the existence of Gauche from isomers of succinic acid have been assumed theoretically, but not confirmed. According to the studies of this inventor, it was found that commercially available succinic acid synthesized by chemical methods is mostly of trans-form and contains a small amount of cis-form. These two may be separated by their solubility diiference in ether. It is also pointed out that there are diiferent shapes of crystals in succinic acid, each being distinguishable under microscopical examination, and these are classified into two types depending on their respective shape at room temperature, i.e. ct-type for needle shape and B-type for hexagonal board or short cylinder. These different crystal types are known as polymorphism and usually has a distinct transition point at which one crystal type can be transformed into the other type. Ordinarily, succinic acid is believed to have no transition point, but the present cis-succinic acid of Gauche form possesses a distinct transition point. As described hereinabove, commercial succinic acid is the mixture of trans-form and a small amount of cis-form (ix-type and B-type). The fi-type crystal is stable at relatively lower temperature while the a-type crystal is stable at relatively higher temperature.

Dupr La Tour reported in Compt. Rend, vol. 193, page 180 (1931) that when ,B-type succinic acid is heated at 138 0., this crystal is converted to wtype and at the same time cis-form acid is produced, and therefore the transition point of succinic acid is 138 C. However, the inventor examined the properties of commercially available succinic acid and found that the trans form succinic acid cannot be converted to the cis-form by mere heating, but can be converted to wtype from B-type when it is first molten at 186 C. and subsequently cooled to precipitate crystals, accordingly, the distinct transition point cannot be observed. On the other hand, it was also found that the pure cis-form succinic acid possesses a considerably clear transition point from pto a-type crystals.

The following important facts were also found by the inventor. That is, the crystal shape of this substance is in general hexagonal board or short cylinder at a room temperature. Upon heating, it is completey sublimed at 147-155 C., but its original shape is still maintained after said sublimation. Continuing the heating, the compound, however, suddenly changes into long cylindrical shape at 156-157 C. From these findings, the inventor ratiocinated that this long cylindrical shape must be the u-type succinic acid.

In the chemical field, it is well known that some chemicals possess optical activity even if they have no asymmetrical carbon atom. For example, dichloroethane having the formula carries no asymmetrical carbon atom, but the intramolecular rotation therein is somewhat restricted by the presence of two chlorine atoms and the free rotation is settled down at positions where the rotation potential is minimal. Thus, the trans-dichloroethane possesses the rotational isomers having one CH Cl turned by around the axis C-C. These rotational isomers are often called Gauche form isomers.

Since succinic acid is represented by either of the following formulae quite similar to that of dichloroethane.

eis-suceinic acid trans-succinic acid Such Gauche form isomers are assumed theoretically, but they have not been confirmed up to the time.

The present inventor has discovered that a Gauche form isomer closely related to the cis-type succinic acid can be prepared together with cis-succinic acid by fermentation with a strain of Saccharomyces genus. These two, i.e. cistype and new Gauche form, are obtained as a mixture, but can hardly be separated from each other because of the similarity in their respective physical and chemical properties. However, this product apparently differs from the existing succinic acids, i.e. trans-form, in many respects as described below, and possesses an outstanding characteristic activity to remove the offensive odor derived from amines. The terms cis-succinic acid of Gauche form used herein refers to the product obtained by the present method.

Accordingly, an object of the present invention is to provide a process for preparing a new and useful compound called cis-succinic acid of Gauche form, particularly by fermentation. Another object of the invention is to provide a method for deodorizing amine-containing materials by contacting them with the said cis-succinic acid of Gauche form. Other objects, features, capabilities and advantages comprehended by the present invention will be apparent from the description and claims which follow.

Referring to the drawings:

FIG. 1 is a curve of the titration of ,B-cis-succinic acid of Gauche form, with N/ 10 sodium hydroxide aqueous solution;

FIG. 2 is a curve of the ultra-violet spectrum of fl-cissuccinic acid of Gauche form; 4

FIG. 3 is a curve of the infra-red spectrum of B-cissuccinic acid of Gauche form; and

FIG. 4 is crystals of fl-cis-succinic acid of Gauche form; and

3 FIG. Sis crystals of a-cis-succinic' acid of Gauche form.

This new compound, cis-succinic acid of Gauche form, can be formed by growing the microorganism, Sacclzaromyces steineri var. [mm in a suitable nutrient medium Growth on malt juice This strain Saccharomyces steineri var. hara shows good vegetative growth on malt juice at 25 C. After three days incubation, oval or elongate cells each having 5 (3.0-7.0) x (5.012.0);L in size are obtained. Usual prounder h? stahhlary m aerobic (vlz' portionof length to width of the cell is 1:2, but some shaken) Conditions Thls fh We have fii f variety is observed. Each cell has the activity of budding floin homemade fermented j and 16 and forms white colored deposits in the culture medium. designate as Saccharomyces stemerz var. hara, is so sirru- N0 film formation is Observei lar in cultural characteristics to a strain of Saccharomyces l0 steineri with minor exceptions that I consider it to be Growth ma]t 1141Ce belonging to the same species with Saccharomyces sleineri. The growth of the Strain on malt agar is just equal to A culture of the living microorganism has been deposited that on malt juice. Them is no i ifi t difference in i h avahahle m the Amencah Type Culture shape and size of cells. The colonies of the strain are COllfiC'tlOlli it has been designated as ATCC No. 16 68. round semi glossy light yellowwhite color creamy The Sham was e h m the fermented grape Julce: After cultivations of the slant culture for one month, and the method of lsolahoh as follows: the colonies are found to be yellowish-white colored,

W grape S Ihlhed and h P a creamy soft semi-glossy and smooth, not wrinkled. sterile flask with cotton-seal. Fermentation was carried Slide l p li is not foumi (An out at for 3 to days; Dhnhg thls Penod, the 20 original strain of Sacch. stefneri, and strains of Sacch. flask wa5 shrred h hwlce a At the end Q cereviciae, Sacch. cereviciae var. ellipsoideus and Sacch. fermehtahoh, a fermehtahoh hquor was taken a P willianus are different from each other in their shape, and hum loohahd Was Inoculated to malt agar P The therefore the present strain can be macroscopically displate was incubated at C. for 48 hours. On the plate tinguished from them.) Surface, the P71116 YP Y Creamy colohles of e dashed 25 Sp0rulati0n.-Spores are readily formed on malt agar. Saechammycee heme ham and the whllei h Usually an ascus contains four round escospores. In this Wrmkled eolomee were e e former eolohles belhg point the present strain is differed from an original strain collected to provide the desired m1croorganism. of Sacch Steiner,-

The present invention will be described with particular reference to the above-mentioned strain of the organism, Fermentation f WW0! but it is to be understood that the fermentative processes The present strain sacchm-omyces Steinerlvar [mm of thls f f embrace not only the i of f shows the following fermentative activity to various sugars. myces sterner: var. lzara but also other Gauche form cis-succinic acid-producing strains of Saccharomyces Glucose genus, such strains being readily produced and isolated by routinely applied isolation and strain-modification Maltese methods which include selection of cultured organisms, Saccharose and exposure of organisms to modifying means such as Lactose X-ray, ultraviolet light, and chemical agents, for example, Ralfinose nitrogen mustards. Illustrative examples of other Gauche 40 form cis-succinic acid-producing strains are Sacchm-a- Assimilation of potassmm "mate myces cercvisiae, Saccharomyces cerevisiae var. ellipso- Potassium nitrate is not assimilated by this strain. idcus and Sacclzaromyces willianus. D

ccOm osztzolto a-butzz Sacc/zaromyces stemerz var. hara (ATCC No. 13668) p f I is characterized by th h i l, cultural, d i This strain does not decompose arbutin- The comparalogical tests set forth in the following table and paraill/e table With respect) the Prhseht Strain and other graphs. assembling known yeast is as follows:

Fermentation Shape of Sediment Ring Pseudothe cell Size of the cell formation formation mycelium Sporulation Glu- Malt- Galaccose 05o tose The present Oval to (thin tree-like. Round, mostly strain. elongate. or no). 4/ascus.

Saccharomyccs Oval to (3-8) x (5-14) (thin) tree-like. Round 2-4/ascus steincri. long-oval. mostly 2.

Sacch. Oval to (2.2-5.5) x 63-14-33) trce- Round or oval willianus. elongate. (3-6.5) x (6.51423),u formation. l 4/ascus. (weak) Sacch. ccrc. Oval to (3.5-9.5) x (6-14-20);l (poor). Round or oval var. long-oval. (2.5-6) x (35-13 1-4/ascus. ollipsoirlcus. (3-7.5) x (5-14) Sacch. Round, short- (5-10) x (0-12) t i (one Round or slightly ccrcvisize. oval or oval. (3-7 x (4.540),; third of the oval 1-4/ascus. (weak (3.5-8) 2; (5-15) strain). or

Sacch. Oval, elongate (2.5-4 )1 (7-26) trec- Round 1-4/ascus. pastorin'nus. 0r sausagcformation.

shapes.

Sacch. carls- Round, short- (5-10) x (513);t usually, Round 1-4/aseus. bcrganisz's. oval or oval. (4-8.5) x (515.5)p. +n(c)longate ce S 0 Saudi. Oval to (3-6) x (6-12) tree-like. Round 1-4/ascus. hctcrogenious. longoval.

Sacch. Oval to (4'8) x (5-15), (primitive). Round 1-4/ascus. t'tulz'cus. long-oval.

Fermentation Sugar assimilation Assimilation Ethanol as sole Splitting of potassium source oi carbon of arbutin Lac- Saccha- Ralfinose Glu- Malt- Galae- Lac- Sacchanitrate tose rose cose ose tose tose rose The present Absent. No growth. Absent. strain.

Saccharomyces Absent. N 0 growth. Absent. stcznerz.

Sncch. Absent. N0 growth or Absent. 'wzllzanus. weak growth.

Sacch. cere. Absent. Usually no growth Absent. var. weak or normal ollipsoidcns. growth.

Secs/ Absent. Usually no growth Absent. cerevzszae. (weak) occasionally weak or normal growth.

Saceh. Absent. No growth. Absent. pastorianns.

Sacch. carls- (OOlIl- Absent. No growth. Absent. bergenisis. plete) Sacch. Absent. No growth. Absent. helerogem'cus.

Sacclz. or Absent. N 0 growth. Absent. italicns. weak The classification and diagnosis are performed based upon The Yeasts a toxonomie study by J. Lodder and N. J. W. Kreger-Van Rij North- Holland Publishing 00., Amsterdam (1952).

As the result of the tests stated, the present strain has been identified as a new strain of Saccharomyces steineri. Analogous strains include Sacch. cereviciae, Sacch. steineri, Sacch. ellipsoideus, Sacch. willianus, etc. In the fermentative activity for raflinose, this strain is similar to Sacch. steinerz', and differed from the strains of other species. This strain for-ms two ascopores in ascus, while the original strain of Sacch. steineri usually forms four ascopores.

In the present invention, grape juice or malt juice may be used as culture medium. Malt juice is rather preferred because it is readily commercially available. Of course other culture media including cane sugar juice, Hennebergs synthetic medium, etc., can be used to produce ,B-cis-succinic acid of Gauche form.

Table 1 shows the result of fermentation tests using shaking culture at 27 C. for 72 hours. A series of these tests was carried out for determining the usefulness of various culture media.

Table 2 shows the result of fermentation tests using standing culture at 27 C. for 14 days. A series of these tests was carried out for determining the usefulness of various culture media.

With respect to cultivation method of the present invention, any of standing culture, shaking culture, submerged aerated culture, etc. may be employed to form 65 /8-cis-succinic acid of Gauche form. However, when aerobic cultivation such as shaking culture and submerged aerated culture is employed, the time required for cultivation is highly minimized. Accordingly, such methods are more preferably used for the purpose of the invention as compared with stationary cultivation methods. Under aerobic conditions, the cultivation may be successfully carried out at about 20 to 30 C. for about 72-96 hours. When stationary conditions are applied to cultivation, it should be carried out at about 20 to 30 C. for about 14-21 days.

TABLE 3.STANDING CULTURE TEST day 7 days 14 days 21 days Medium Tslp Sugar pI-I Sugar pH Sugar p11 Sugar pH Percent Percent Percent Percent Grape juice 27 30 14. 6 3. i=7. 9 3.0 +6. 4 2. 7 ++4. 6 2. 5 -20 14. 6 3. 5 8. 2 3. 2 3:7 2 3.0 +6. 6 2.3 5 14. 6 3. 5 9. 4 3. 4 i7. 6 3. 2 +7. 4 3.0 Malt juice 27-30 13. 2 5. O 8. 6 3. 4 +5. 4 3. 0 ++3. 2 2. 7 15-20 13. 2 5. 0 0. 6 3. 8 5:7. 4 3. 2 +4. 0 2. 9 5 13. 2 5. 0 11. 4 4. G 10. 0 4.0 18.0 3. 8

TABLE 4.SHAKING CULTURE TEST 0 hr. 24 hrs. 48 hrs. 72 hrs. Medium T enp Sugar pH Sugar p11 Sugar pH Sugar p11 Percent Percent Percent Percent Grape juice 27 15.0 3. 5 5:12. 0 2. 5 ++5. 0 2. 5 ++4. 6 2. 5 Malt juice 27 15.0 5.0 i5. 4 3. 7 ++5. 2 3. 4 ++4. 2 3. 2

TABLE 5.-COMPARATIVE TEST FOR SI-IAKIN G CULTURE USING VARIOUS STRAINS 0 hr. 12 hrs. 24 hrs. 48 hrs. 72l11s. Strain Sugar pH Sugar pH Sugar ph Sugar pH Sugar pH Saceh. steineri var. Percent Percent Percent Percent Percent haran' 13 5.0 2 4. 6 11:5. 4 3. 7 ++3. 1 3.1 ++2. 8 3. 0 Sacch. steineri 13 5.0 -12.9 4. 8 5:6. 5 4.0 +4.0 3.8 ++4. 0 3.8 Sacch. c.v. ellipsoideus 13 5.0 12. 8 4.8 $6. 6 3. 9 +4. 6 3.9 ++4. 0 3. 8 Sacch. cerevieiae 13 5.0 12. 9 4. 9 10.1 4.0 10.0 4.0 5:10. 0 4. 2

In Tables 3 and 4, both tests were carried out using a strain Saccharomyces steineri var. ham (ATCC No. 13668). In Table 5, the tests were carried out at 27 C., using malt juice medium. In each of the above tables, signs (1), and indicate the degree of deodorization effect.

In carrying out the invention in effect, the culture solution obtained by cultivation stated above is extracted with ether, and then the ether is removed from the extract. The remaining part is further concentrated to yield crude crystal of Gauche form fi-cis-succinic acid. The crude product can be recrystallized from hot ether to give pure crystal.

Thus, the Gauche form succinic acid can be prepared economically by the fermentation with a strain Saccharomyces steineri var. hara (ATCC No. 13668) and by an ether extraction of the culture solution obtained. The inventor has further discovered that the above said ether extract contains a useful enzyme like yellow substance, that is, the water solution of commercially obtainable succinic acid and maleic acid were changed to the B-cis-succinic acid of Gauche form by contacting with above yellow substance under light shielding conditions.

Firstly an enzyme like substance containing liquor is prepared by evaporating the solvent from the above said ether extract. A mixture of equal amounts of known, commercially available succinic acid (optical isomer) and maleic acid or maleic anhydride is dissolved in twice the amount of distilled water under heating, After cooling to a temperature below 50 C., the resulting solution is combined with the enzyme like substance containing liquor under day-light shielding conditions. The resulting mixture is concentrated under reduced pressure to yield crude crystal of ,B-cis-succinic acid of Gauche form.

According to the method described herein, almost 90% of the total weight of the succinic acid and maleic acid employed can be converted to the Gauche form succinic acid.

With respect to the production of fi-cis-succinic acid of Gauche form, typical procedures used for carrying out the invention will be described in the following.

Two hundreds milliliters of malt juice was charged into a 500 cc.-volume Sakaguchi flask and then sterilized with heat at C. for 20 minutes. After cooling, a strain Sacch. steineri var. hara was planted to the flask. Before cultivation, malt juice preferably had a pH of 4.6 to 5.6 and a sugar content of 15 to 17%. Cultivation temperature was 25 15 C. and cultivation period was 72 to 120 hours. During cultivation, the flask was shaken by means of a reciprocating type shaking machine which was reciprocated 120 times per minute with an amplitude of 8 cm. After cultivation, the culture solution was found to have a pH of 2.8 to 3.2 and a sugar content of 3 to 4%.

Extraction of fl-cis-succinic acid of Gauche form from culture solution From the culture solution obtained by cultivation stated, mycelia were removed by filtration. The filtrate, charged into an automatic liquid extractor, was extracted with ether over 36 to 48 hours. An other fraction was recovered, and the ether was removed under reduced pressure. The remaining part was further concentrated whereby crude crystal of ,B-cis-succinic acid of Gauche form was yielded. This crude crystal had yellow color and characteristic fermentative odor. From one liter of culture solution, about one gram of crude crystal was obtained.

The crude crystal thus obtained was dissolved into a small amount of hot ether, and filtrate with small amount of carbon powder, and then cooled in order to recrystallization. Repeating recrystallization, about 500 mg. of pure crystal were obtained from about one gram of the crude one.

Similarly fi-cis-succinic acid of Gauche form was extracted from a culture solution which was obtained by cultivation of the other strains of Saccharomyces genus than Sacch. steineri var. hara such as referred to above. In this case, to 400 mg. of pure crystalwas recovered from one liter of the culture solution.

Extraction of an enzyme like subst. containing liquor from the culture solution From the culture solution, mycelia were removed by filtration. By means of an automatic liquid extractor, the

commercial succinic acid had no sublimation at the same temperature.

Gauche form succinic acid: (.B-type crystal) sublime (anhydride; B-type crystal) filtrate was extracted with ether for 36 to 48 hours. An 147 0. 156 0. ether fraction was recovered, and the ether was removed (tr-type crystal) (melt at 196197 0.) under reduced pressure. The remaining part was a brown colored liquid containing fl-cis-Succinic acid of Gauche Commercial succinic acid; (t pe crystal) form together with the yeast used. This liquor (including don't sublime cool the said yellow substance) was recovered in an amount (melt at 1860 (Hype Hymn of about 5 cc. from one liter of the culture solution. Determination molecular 8 Syn thetic procedures using the enzyme like yellow sub- Molecular Weight of Substance Was determined smncewonmmiilg liquor cording to Akiya and Bergers method. Alcohol was used as solvent and azobenzene as known control. As the reg g'giz f i fi gi g ig s g g g 323x 2 ggg s gfig sult, it was found that the molecular weight of the subc l dride) was dissolved in twice amount of distilled water or Stance was approxlmately of less than 50% ethanol solution with heat. After cooling Elementary mall/SIS below the resulting F was combmed w 1th Elementary analysis of this substance showed that the the above extracted enzyme like yellow substance which 29 Substance contained 40 83% of Carbon, 5 14% of hydro was in Such amount as Correspondmg to more than gen and 54.03% of oxygen, but not nitrogen and halogen. by weight of the said solution. While shielding from day See Table light, the resulting mixture was concentrated under re- TABLE 6 ELEMENTARY ANALYSIS duced pressure of less than 20 mm. 'Hg, whereby crude crystal of fl-cis-succinic acid of Gauche form was yielded. O H N: N The crude crystal could be recrystallized in the same manner mentioned above' Percent Percent Cc. Percent It should be noted that any other solvent than ether Th prcsentsubstan e. 40.83 5.14 0 0 cannot be used for the recovery of Gauche form cis- 40.68 12 0 0 succinic acid from the culture solution because such sol- Maleic a i 41.39 3.47 0 0 vent cannot dissolve selectively the desired substances, and in the present, therefore, ether should be critically Acid titration and the curve thereof used for the extraction of the substances.

An aqueous solution of this substance was titrated with clsisuctclmlc i g fi l i s N/lO-NaOH aqueous solution, and as its result the moj arac ellze g fi f erinca an lecular weight was found to be about 119. Further such p yslo Oglca propemes set oft e O owmg paratitration was repeated using an electric automatic titra graphs M l tion apparatus, and the data were plotted on a titration e zmg pomt curve. The present substance gave the similar titration Melting points of the present compound and the com- 40 curve to that of known succinic acid, and was found to mercial succinic acid were determined macroscopically as be dicarboxylic acid. '(See FIG. 1.) follows:

Sample was sealed in a capillary and heated compara- Qualmtlve test of ca'bonyl group tively lapid in sulfuric acid bath. The precipitation reaction using 2,4-dinitrophenyl hy- C, drazine was carried out for determining whether carbonyl The present compound (5 form) M.P. 183 to 184 group was present or not in the substance. It was found Succinic acid, commercially available 185 to 186 that the present substance had not C=O group. Mixed sample of the above two 184 to 185 Qualitative test of hydmxyl group a melting pomt of the present compound was As the result of diazo color reaction, it was found that determined by micro method, and the following changes the present Substance did not contain hydroxyl group. were observed: I I l Id bl b d Room temp. (hexagonal board or short cylinder shaped Qua f f testof Gilly 6mm e on Crystal) The decolorizatlon reaction of an aqueous solution of Sub1imation point (147 156 Q) (Crystal shape this substance by a potass um permanganate aqueous soluchanged; 4 m tron, and the decolorizatlon reaction of a glacial acetic or above (Needle Shaped crystal; wfol-m) acid solution of this substance by a glacial acetic acid Me1ting 7 Q) solut on of bromine were tested, and in both cases dea o colonzation was not observed. It was found that this sub- In the above changes the point of 156 l57 C. seemed stance had not an ethylenical double bond (C C), to be the metastasis polnt (transition point) ({i-succrnlc 60 acid to u-SUCCllllC acid). But Dupr La Tour said that the Solublllty to Solvents trfmsitioi} POint 0b5eYVed is This substance has a relatively lower solubility in wa- Thus, In a mum-method p y g a small amount of ter, ethanol or ether, than that of a known, commercially fine crystals and heating very carefully, all of the crystals available succinic acid. Also this substance is slightly soltested were sublimated at a temperature below 156 C. 65 uble in benzene and chloroform, and unsoluble in petroto give anhydride of Gauche form succinic acid, while the leurn ether and carbon tetrachloride.

Solvents n Water l Ethanol Methanol Ether chloroform Substances:

Present substance 25.0 ml 20.0 10.0 200 Non. Succinic acid 13.0 18.5 6.3 113 N011. Maleic acid Freely Freely Freely Slightly Funnaric acid 158.7 52. 32. Non.

1 1 (One gram of samples were dissolved'at 20 C. and one atmosphere pressure.)

Specific rota-tion This substance (ti-form) and the polymorph thereof and a known, commercially available succinic acid were measured of their specific rotation:

The present substance (Ii-form) (1% aq. soln.) 18.1

(oi-form) (1% aq. soln.) 18.4 Commercially available succinic acid (1% aq. soln.) -1.7

The brucine salt of this substance reacted at room tem perature and that of a known succinic acid were meas-- ured of their specific rotation.

Brucine saltof this substance (1% aq. soln.) 19.3 Brucine succinate, commercially available (1% aq. soln.) 32.3

U lira-violet absorption In this test, an aqueous solution of this substance (1 mg./ 10 cc.) was used as sample. For contrast, known succinic acid, maleic acid and fumaric acid were employed. As shown in FIG.-2, the ultra-violet absorption spectrum of the substance shows only one maximum at 200 m while those of succinic, maleic and fnmaric acids. show one maximum at 210 m 212 m and 208 m respectively.

Infra-red absorption The infra-red absorption spectrum of this substance is entirely similar to that of a known, commercially available succinic acid, as seen in FIG. 3.

Anhydride of fl-cis-succinic acid of Gauche form The substance (fl-cis-succinic acid of Gauche form) is completely Sublimated at temperatures between 147 and 156 C. thereby to convert it into its anhydrous form. Such anhydrous substance is characterized in that the infra-red absorption spectrum thereof shows absorption bands at wave length: 1780 and 1875 mf This anhydrous substance when dissolved into water, immediately is converted into dicarboxylic acid by ring opening.

Reactivity test with IhI"C0mPOLlndS When a 1 N aqueous solution of this substance is added with a 1 N ammonia solution, a chemical reaction takes place immediately at room temperature whereby asparagine and aspartic acid are formed. Commercially available succinic acid does not react with ammonia at a room temperature.

Rf values on paper chromatogram Thefollowing data show the Rf values of the substance and other various known organic acids, which are determined by one dimensional paper-chromatogram.

In each of the runs, the development was carried out at 20 C. for 15 hours, using Toyo filter paper No. 50 or 51 and bromo cresol green as coloring agent.

Physiological activity of this substance When fresh fish meat, whale meat, bird and animal meat, and the internal organs thereof are immersed into a 0.1 to 0.05% aqeuous solution of this substance, the peculiar offensive odor disappears after immersion at an ordinary temperature for 2 to 6 hours or at 0 to 5 C. for 24 to 96 hours.

Fish or animal meat is digested withproteinase, and then indigestible residue and fat and fatty oils are removed therefrom. The resulting solution is adjusted with a 5 N KOH aqueous solution at a pH of 7.0 to 7.4, and this substance is added in an amount of 0.1 to 0.05% to them. While maintained at to 100 C. for 60 minutes, the

solution is readjusted at a pH of 7.6 to 8.0. After filtra-.

tion, the filtrate is adjusted at a pH of 5.0 with 5 N HCl, and then concentrated to about one-fifth volume. The concentrate is added with about 1 or 2% starch, and then sprayor freeze-dried, whereby a deodorized powder containing a large amount of essential amino acids is obtained. Such powder is suited for the use of food.

A crude liquor removed of sodium glutamate crystals is adjusted at a pH of 7.0 to 7.2 by addition of 5 N KOH, and then heated up to 70 C. The liquor is added with 0.1 to 0.05% of the present substance, allowed to stand for 30 to 60 minutes, and then blown with sterile air for. 10 to 20 minutes. Volatile, odoriferous materials mainly including dimethyl sulfite are liberated and purged away. The liquor thus treated is adjusted again at a pH of 7.6 to 8.0 witha KOH aqeuous solution and then filtered. Black sediment is removed. The filtrate is adjusted at a pH of 4.6 to 4.7 with5 N HCl. Thus a deodorized solution removed of sodium glutamate crystals (or amino acids) is obtained. This liquid is quite suitable to soysauce additive.

Mucous odor and putrefied tissue odor are removed by washing the area with a 0.1-0.05% aqueous solution of this substance. Also toilet odor can be killed by addition of the said solution. The smell of armpit is prevented by applying an ointment containing 0.1 to 1.0% of this substance.

Toxic amines such asyhistamine, indole and scatole, when reacted with the substance, are converted to such compounds which are non-toxic and odorless.

The mechanism of the deodorizing action of the present compound may be explained as follows:

C-COOII /CCOOH H 4 H I A t 'dt.

unnc s ar icaci e a yr CGOOII C-COOH H l Amine C-CO-Amme l H A t s aragm e p by C-COOH Ammo That is, when meat or fish is hydrolyzed, various kinds of amino-acids can be produced, but the hydrolysis solution may contain various amines as a by-product, which causes a characteristic odor of the meat or fish.

The Gauche form succinic acid can be easily reacted with these free amines at a room temperature to give aspartic acid typeor asparagin type products thereby inhibiting a characteristic deodorizing action. Such an action can not be found in the commercially available succinic acid.

What I claim is:

1. A process for producing cis-succinic acid of Gauche form, which comprises cultivating a strain of Saccharomyces genus in an aqueous, malt juice-containing solution under aerobic conditions at a temperature of from about 20 to about 35 C. for 72-120 hours, extracting cis-succinic acid of Gauche form With ether under day light shielding conditions, and distilling the ether extract in vacuo while shielding it from day light to give the crystals of cis-succinic acid of Gauche form.

2. A process according to claim -1, wherein said strain is selected from the group consisting of the strains of Sacch. steineri species, and Sacch. cereviciae ellipsoideus species.

3. A process for producing cis-succinic acid of Gauche form, which comprises cultivating a strain of Saccharomyes steineri var. hara ATCC No. 13688 in aqueous, malt juice-containing solution under aerobic conditions 14 at a temperature of from about 20 to about 35 C. for 72120 hours, extracting cis-succinic acid of Gauche form with ether under day light shielding conditions, and distilling the ether extract in vacuo while shielding it from day light to give the crystal of cis-succinic acid of Gauche form.

4. A process for producing cis-succinic acid of Gauche form, which comprises cultivating a strain of Saccharomyces steineri var. ham ATCC No. 13668 in an aqueous, malt-juice-containing solution under aerobic conditions at about 20-35 C. for 72-120 hours, extracting cissuccinic acid of Gauche form With ether under day light shielding conditions, distilling otf the solvent, adding with an aqueous solution of commercial succinic acid and a member selected from the group consisting of commercial maleic acid and commercial maleic anhydride, and distilling oil water in vacuo to give a langer quantity of cis-succinic acid of Gauche form.

5. A process according to claim 4, wherein said cissuccinic acid of Gauche form is further purified by dissolving it in ether, treating with active charcoal and distilling oh the solvent, under day light shielding conditions.

No references cited.

A. LOUIS MONACELL, Primary Examiner.

L. M. SHAPIRO, Assistant Examiner. 

1. A PROCESS FOR PRODUCING CIS-SUCCINIC ACID OF GAUCHE FORM, WHICH COMPRISES CULTIVATING A STRAIN OF SACCHAROMYCES GENUS IN AN AQUEOUS, MALT JUICE-CONTAINING SOLUTION UNDER AEROBIC CONDITIONS AT A TEMPERATURE OF FROM ABOUT 20* TO ABOUT 35*C. FOR 75-120 HOURS, EXTRACTING CIS-SUCCINIC ACID OF GAUCHE FORM WITH ETHER UNDER DAY LIGHT SHIELDING CONDITIONS, AND DISTILLING THE ETHER EXTRACT IN VACUO WHILE SHIELDING IT FROM DAY LIGHT TO GIVE THE CRYSTALS OF CIS-SUCCINIC ACID OF GUACHE FORM. 